The greatest challenge of the post genomic era is to uncover the functions of every individual human gene. Development of novel high throughput functional genomics tools is critical for a genome-wide discovery of a gene's functions and for linking their products into signaling pathways. The recent introduction of gene knockdown technology, based on small interfering RNAs (siRNA), promises to revolutionize gene functional analysis. Introduction of selectable pathwayspecific reporter systems would significantly complement the siRNA approach that is aimed at functional dissection of normal and disease-affected pathways. The goal of the proposed program is to develop and make commercially available a set of safe lentiviral transcriptional reporter vectors for the most critical disease-related signal transduction pathways. These pathway-specific reporter vectors, in combination with gene-specific siRNAs or genome-wide siRNA libraries, will allow researchers to study the mechanisms of pathways and identify changes in the mechanisms involved in the pathogenesis of human diseases. Comprehensive genetic screens with genome-wide siRNA libraries and pathwayspecific reporter vectors could identify target genes whose inactivation leads to suppression of disease-related changes. Phase I of the program will cover the development of technology for construction of (i) next-generation safe lentiviral-based reporter constructs for detection of changes within p53 pathway, and (ii) lentiviral siRNA-expressing vectors designed for silencing of several known components of the p53 pathway. Functional performance of these vectors will be tested in normal (human embryonic fibroblasts, HEFs) and disease (cervical carcinoma cell line HeLa) model systems. The Phase II of the program will be extended towards development and commercialization of a comprehensive set of lentiviral reporter vectors with beta-galactosidase, luciferase and fluorescent reporters, covering about 30 major signal transduction pathways associated with a wide range of human diseases. The developed dual color fluorescent reporter vectors and genome-wide siRNA libraries, comprising all known genes, will allow a global search for novel pathway-specific components and disease-associated genes in the context of living cells. The established technology will be applied, in collaboration with the Cleveland Clinic Foundation, for identification of targets for drug development aimed at the restoration of suppressed p53 pathway in a set of human carcinoma models. The anticipated outcomes of the proposed research and product development program will be a commercially available set of kits comprising of lentiviral reporter vectors and siRNA libraries for high-throughput gene functional analysis, custom genome-wide functional screen service and several validated anticancer drug targets.